FUNCTIONAL ROLE OF PI3K-DELTA IN MALIGNANT B CELLS
(Abstract release date: 05/19/16)
EHA Library. Neumann L. 06/09/16; 132593; E1044
Dr. Lars Neumann
Contributions
Contributions
Abstract
Abstract: E1044
Type: Eposter Presentation
Background
Among catalytic PI3K subunit isoforms, p110-δ is highly and selectively expressed in malignant compared to other cell types and involved in B cell receptor (BCR) signaling. Idelalisib, which specifically targets p110-δ, shows clinical efficacy e.g. for the treatment of chronic lymphocytic leukemia (CLL), but only modest direct cytotoxicity against malignant B cells in vitro.
Aims
To elucidate the mechanism of action of idelalisib and to dissect the contributions of p110-δ to both cell autonomous functions as well as interactions with the tumor micro-environment, engineered inhibitor-resistant p110-δ mutants were employed as chemical genetic tools. In addition cell line models expressing such mutants might anticipate a potential type of clinically acquired resistance.
Methods
According to structural alignment with known inhibitor-resistant p110-α mutants, p110-δ-I777M and –I825V were generated by site-directed mutagenesis as potentially inhibitor-resistant mutants. Inhibitor resistance was assessed by comparing phospho-Akt levels after inhibitor treatment in retrovirally transduced cell lines expressing wild type or mutant p110-δ. Transductants of the murine fibroblast cell line NIH3T3 were assessed for anchorage-independent growth and saturation density as markers of oncogenic transformation. The emphasis of our chemical genetic evaluation was on malignant B cells and employed the human Burkitt lymphoma and murine pre-B cell lines Ramos and BaF/3. Chemokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay, while chemotaxis to CXCL12 was examined in trans-well migration assays.
Results
The murine fibroblast cell line NIH 3T3, which lacks endogenous p110-δ expression and sensitivity to idelalisib, gained sensitivity to idelalisib concentrations above 1 µM upon over-expression of p110-δ with regard to phosphorylation of Akt at serine 473. According to pAkt levels, the I777M mutation in p110-δ partly reversed the sensitivity to idelalisib of NIH 3T3 cells expressing p110-δ. Moreover expression of p110-δ-I777M increased the anchorage-independent growth and the saturation density of NIH3T3 cells. In Ramos and Baf-3 cells, which endogenously express p110-δ, additional expression of p110-δ-I777M led to markedly increased Akt phosphorylation and to resistance against 10-100 nM idelalisib. Expression of p110-δ-I777M rescued the secretion of the chemokine CCL-3 by Ramos cells upon stimulation of the B-cell receptor from inhibition by idelalisib. Chemotaxis of Baf-3 cells to the chemokine CXCL-12 was reconstituted by expression of p110-δ-I777M in the presence of inhibitory concentrations of idelalisib.
Conclusion
The novel engineered p110-δ-I777M mutant showed an unexpected gain of function and mediated resistance against idelalisib that can be exploited for the chemical genetic evaluation of p110-δ function. In malignant B cell lines, p110-δ was less involved in cell autonomous functions than in their interactions with the micro-environment, exemplified by chemokine secretion and migration.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Kinase inhibitor, PI3K
Type: Eposter Presentation
Background
Among catalytic PI3K subunit isoforms, p110-δ is highly and selectively expressed in malignant compared to other cell types and involved in B cell receptor (BCR) signaling. Idelalisib, which specifically targets p110-δ, shows clinical efficacy e.g. for the treatment of chronic lymphocytic leukemia (CLL), but only modest direct cytotoxicity against malignant B cells in vitro.
Aims
To elucidate the mechanism of action of idelalisib and to dissect the contributions of p110-δ to both cell autonomous functions as well as interactions with the tumor micro-environment, engineered inhibitor-resistant p110-δ mutants were employed as chemical genetic tools. In addition cell line models expressing such mutants might anticipate a potential type of clinically acquired resistance.
Methods
According to structural alignment with known inhibitor-resistant p110-α mutants, p110-δ-I777M and –I825V were generated by site-directed mutagenesis as potentially inhibitor-resistant mutants. Inhibitor resistance was assessed by comparing phospho-Akt levels after inhibitor treatment in retrovirally transduced cell lines expressing wild type or mutant p110-δ. Transductants of the murine fibroblast cell line NIH3T3 were assessed for anchorage-independent growth and saturation density as markers of oncogenic transformation. The emphasis of our chemical genetic evaluation was on malignant B cells and employed the human Burkitt lymphoma and murine pre-B cell lines Ramos and BaF/3. Chemokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay, while chemotaxis to CXCL12 was examined in trans-well migration assays.
Results
The murine fibroblast cell line NIH 3T3, which lacks endogenous p110-δ expression and sensitivity to idelalisib, gained sensitivity to idelalisib concentrations above 1 µM upon over-expression of p110-δ with regard to phosphorylation of Akt at serine 473. According to pAkt levels, the I777M mutation in p110-δ partly reversed the sensitivity to idelalisib of NIH 3T3 cells expressing p110-δ. Moreover expression of p110-δ-I777M increased the anchorage-independent growth and the saturation density of NIH3T3 cells. In Ramos and Baf-3 cells, which endogenously express p110-δ, additional expression of p110-δ-I777M led to markedly increased Akt phosphorylation and to resistance against 10-100 nM idelalisib. Expression of p110-δ-I777M rescued the secretion of the chemokine CCL-3 by Ramos cells upon stimulation of the B-cell receptor from inhibition by idelalisib. Chemotaxis of Baf-3 cells to the chemokine CXCL-12 was reconstituted by expression of p110-δ-I777M in the presence of inhibitory concentrations of idelalisib.
Conclusion
The novel engineered p110-δ-I777M mutant showed an unexpected gain of function and mediated resistance against idelalisib that can be exploited for the chemical genetic evaluation of p110-δ function. In malignant B cell lines, p110-δ was less involved in cell autonomous functions than in their interactions with the micro-environment, exemplified by chemokine secretion and migration.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Kinase inhibitor, PI3K
Abstract: E1044
Type: Eposter Presentation
Background
Among catalytic PI3K subunit isoforms, p110-δ is highly and selectively expressed in malignant compared to other cell types and involved in B cell receptor (BCR) signaling. Idelalisib, which specifically targets p110-δ, shows clinical efficacy e.g. for the treatment of chronic lymphocytic leukemia (CLL), but only modest direct cytotoxicity against malignant B cells in vitro.
Aims
To elucidate the mechanism of action of idelalisib and to dissect the contributions of p110-δ to both cell autonomous functions as well as interactions with the tumor micro-environment, engineered inhibitor-resistant p110-δ mutants were employed as chemical genetic tools. In addition cell line models expressing such mutants might anticipate a potential type of clinically acquired resistance.
Methods
According to structural alignment with known inhibitor-resistant p110-α mutants, p110-δ-I777M and –I825V were generated by site-directed mutagenesis as potentially inhibitor-resistant mutants. Inhibitor resistance was assessed by comparing phospho-Akt levels after inhibitor treatment in retrovirally transduced cell lines expressing wild type or mutant p110-δ. Transductants of the murine fibroblast cell line NIH3T3 were assessed for anchorage-independent growth and saturation density as markers of oncogenic transformation. The emphasis of our chemical genetic evaluation was on malignant B cells and employed the human Burkitt lymphoma and murine pre-B cell lines Ramos and BaF/3. Chemokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay, while chemotaxis to CXCL12 was examined in trans-well migration assays.
Results
The murine fibroblast cell line NIH 3T3, which lacks endogenous p110-δ expression and sensitivity to idelalisib, gained sensitivity to idelalisib concentrations above 1 µM upon over-expression of p110-δ with regard to phosphorylation of Akt at serine 473. According to pAkt levels, the I777M mutation in p110-δ partly reversed the sensitivity to idelalisib of NIH 3T3 cells expressing p110-δ. Moreover expression of p110-δ-I777M increased the anchorage-independent growth and the saturation density of NIH3T3 cells. In Ramos and Baf-3 cells, which endogenously express p110-δ, additional expression of p110-δ-I777M led to markedly increased Akt phosphorylation and to resistance against 10-100 nM idelalisib. Expression of p110-δ-I777M rescued the secretion of the chemokine CCL-3 by Ramos cells upon stimulation of the B-cell receptor from inhibition by idelalisib. Chemotaxis of Baf-3 cells to the chemokine CXCL-12 was reconstituted by expression of p110-δ-I777M in the presence of inhibitory concentrations of idelalisib.
Conclusion
The novel engineered p110-δ-I777M mutant showed an unexpected gain of function and mediated resistance against idelalisib that can be exploited for the chemical genetic evaluation of p110-δ function. In malignant B cell lines, p110-δ was less involved in cell autonomous functions than in their interactions with the micro-environment, exemplified by chemokine secretion and migration.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Kinase inhibitor, PI3K
Type: Eposter Presentation
Background
Among catalytic PI3K subunit isoforms, p110-δ is highly and selectively expressed in malignant compared to other cell types and involved in B cell receptor (BCR) signaling. Idelalisib, which specifically targets p110-δ, shows clinical efficacy e.g. for the treatment of chronic lymphocytic leukemia (CLL), but only modest direct cytotoxicity against malignant B cells in vitro.
Aims
To elucidate the mechanism of action of idelalisib and to dissect the contributions of p110-δ to both cell autonomous functions as well as interactions with the tumor micro-environment, engineered inhibitor-resistant p110-δ mutants were employed as chemical genetic tools. In addition cell line models expressing such mutants might anticipate a potential type of clinically acquired resistance.
Methods
According to structural alignment with known inhibitor-resistant p110-α mutants, p110-δ-I777M and –I825V were generated by site-directed mutagenesis as potentially inhibitor-resistant mutants. Inhibitor resistance was assessed by comparing phospho-Akt levels after inhibitor treatment in retrovirally transduced cell lines expressing wild type or mutant p110-δ. Transductants of the murine fibroblast cell line NIH3T3 were assessed for anchorage-independent growth and saturation density as markers of oncogenic transformation. The emphasis of our chemical genetic evaluation was on malignant B cells and employed the human Burkitt lymphoma and murine pre-B cell lines Ramos and BaF/3. Chemokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay, while chemotaxis to CXCL12 was examined in trans-well migration assays.
Results
The murine fibroblast cell line NIH 3T3, which lacks endogenous p110-δ expression and sensitivity to idelalisib, gained sensitivity to idelalisib concentrations above 1 µM upon over-expression of p110-δ with regard to phosphorylation of Akt at serine 473. According to pAkt levels, the I777M mutation in p110-δ partly reversed the sensitivity to idelalisib of NIH 3T3 cells expressing p110-δ. Moreover expression of p110-δ-I777M increased the anchorage-independent growth and the saturation density of NIH3T3 cells. In Ramos and Baf-3 cells, which endogenously express p110-δ, additional expression of p110-δ-I777M led to markedly increased Akt phosphorylation and to resistance against 10-100 nM idelalisib. Expression of p110-δ-I777M rescued the secretion of the chemokine CCL-3 by Ramos cells upon stimulation of the B-cell receptor from inhibition by idelalisib. Chemotaxis of Baf-3 cells to the chemokine CXCL-12 was reconstituted by expression of p110-δ-I777M in the presence of inhibitory concentrations of idelalisib.
Conclusion
The novel engineered p110-δ-I777M mutant showed an unexpected gain of function and mediated resistance against idelalisib that can be exploited for the chemical genetic evaluation of p110-δ function. In malignant B cell lines, p110-δ was less involved in cell autonomous functions than in their interactions with the micro-environment, exemplified by chemokine secretion and migration.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Kinase inhibitor, PI3K
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